Flatbush is the neighborhood where Mario and Luigi grew up and is also where they set up Mario Brothers Plumbing. As such, all the live-action segments take place in Flatbush. A famous street in Flatbush is the eponymous named Flatbush Avenue. Under the orders of Mama Mario, Mario once "washed [the street] as far as Flatbush Avenue."

During return trips to Brooklyn, it is unknown if the neighborhood of Flatbush is actually visited or not. Salvador Drainado claimed to be from Flatbush and managed to return home after being stuck on Mushroom World for thirteen years in "Brooklyn Bound".

In "Flatbush Koopa", King Koopa, after forcing the Koopa Pack to disband in the Mushroom Kingdom, attempted to take over all of Brooklyn. Upon seeing Mario and Luigi, he threatened to flatten them worse than he was going to flatten Flatbush. However, his takeover was ultimately foiled.